Quantum dots as nanolabels for breast cancer biomarker HER2-ECD analysis in human serum

Talanta 208 (2020) 120430

Contents lists available at ScienceDirect

Talanta journal homepage: www.elsevier.com/locate/talanta

Quantum dots as nanolabels for breast cancer biomarker HER2-ECD analysis in human serum

T

Maria Freitas, Marta M.P.S. Neves, Henri P.A. Nouws∗, Cristina Delerue-Matos REQUIMTE/LAQV, Instituto Superior de Engenharia do Porto, Politécnico do Porto, Rua Dr. António Bernardino de Almeida 431, 4200-072, Porto, Portugal

ARTICLE INFO

ABSTRACT

Keywords: Electrochemical immunosensor Breast cancer HER2-ECD Quantum dots Screen-printed electrodes

Early detection of cancer increases the possibility for an adequate and successful treatment of the disease. Therefore, in this work, a disposable electrochemical immunosensor for the front-line detection of the ExtraCellular Domain of the Human Epidermal growth factor Receptor 2 (HER2-ECD), a breast cancer biomarker, in a simple and efficient manner is presented. Bare screen-printed carbon electrodes were selected as the transducer onto which a sandwich immunoassay was developed. The affinity process was detected through the use of an electroactive label, core/shell CdSe@ZnS Quantum Dots, by differential pulse anodic stripping voltammetry in a total time assay of 2 h, with an actual hands-on time of less than 30 min. The proposed immunosensor responded linearly to HER2-ECD concentration within a wide range (10–150 ng/mL), showing acceptable precision and a limit of detection (2.1 ng/mL, corresponding to a detected amount (sample volume = 40 μL) of 1.18 fmol) which is about 7 times lower than the established cut-off value (15 ng/mL). The usefulness of the developed methodology was tested through the analysis of spiked human serum samples. The reliability of the presented biosensor for the selective screening of HER2-ECD was confirmed by analysing another breast cancer biomarker (CA15-3) and several human serum proteins.

1. Introduction The incidence of cancer has increased considerably worldwide, and affects the general population, resulting in significant mortality rates [1,2]. Screening and early detection allow appropriate treatments according to the stage of the disease and the availability of healthcare resources. Most European countries have national cancer screening programmes for colorectal [3,4], cervical [5,6] and breast [7,8] cancer. Breast cancer is an important public health concern with considerable impact for the patients, families and society as a whole. Its incidence and prevalence reveals slight differences among developed and developing countries, with the former presenting a global reduction in mortality due to a combination of improvements in prevention, detection and treatment [1,9]. In fact, some studies indicate that mortality rates have been diminishing in places where active screening programmes (e.g. mammography) were implemented [7–10]. Therefore, new analytical tools for the point-of-care (POC) detection of this disease at the early stages, as well as during its management and follow-up, are widely demanded. Moreover, the development of portable equipment for in situ breast cancer analysis could also be very useful in less developed regions, remote access areas or even for patients with reduced mobility. ∗

The guidelines established by the European Group on Tumor Markers (EGTM) on the use of breast cancer biomarkers for decisionmaking regarding the treatments to be administered were recently updated. Despite the large number of new biomarkers that have been reported, only three are mandatory for all patients diagnosed with invasive breast cancer: oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) [11]. The latter is established as an important diagnostic biomarker and is recommended for testing since this protein is overexpressed in 15–20% of primary invasive breast cancers and is related with the most aggressive phenotypes [12,13]. HER2 is a transmembrane protein located on the cell surface and has distinct domains: an extracellular domain (ECD), an intracellular tyrosine kinase domain and a transmembrane lipophilic segment. ECD can be cleaved by metalloproteases and released into the bloodstream. Consequently, HER2 can be measured in serum, which is key for the development of quantitative analytical strategies for breast cancer detection [14,15]. Currently, the two main types of tests accepted for the evaluation of HER2 overexpression in clinical use are immunohistochemistry and in situ hybridisation (ISH), which are not suited for POC diagnostics [11]. Hence, a considerable effort in the development of new alternative methodologies has been carried out. The use of “lab-on-a-chip” and

Corresponding author. E-mail address: [email protected] (H.P.A. Nouws).

https://doi.org/10.1016/j.talanta.2019.120430 Received 14 June 2019; Received in revised form 24 September 2019; Accepted 1 October 2019 Available online 03 October 2019 0039-9140/ © 2019 Elsevier B.V. All rights reserved.

Talanta 208 (2020) 120430

M. Freitas, et al.

biosensor technologies are currently providing remarkable tools for POC analysis. The possibility of miniaturization, portability, fast response and low cost are some of the electrochemical biosensors' features that make them attractive for the development of new analytical devices [16,17]. Electrochemical-based approaches for the detection of HER2-positive breast cancer have already been proposed. Different biosensing strategies were reported, which include nanomaterial-based sensing- and/or detection platforms [18–24], nanoelectrode ensembles (NEEs) [25], magnetic-based immunoassays [26,27], a sandwich-type immunoassay based on nanobodies [28], sensing strategies based on affibody/antibody recognition events [29], a MIP-based sensor [30], an impedimetric biosensor based on a zwitterionic hydrogel [31], an impedimetric immunosensor based on the use of single-chain fragment variable antibody fragments [32], a cellulase-linked sandwich assay [33], an inkjet-printed electrochemical platform [34], a polycytosine DNA-based immunosensor [35], and a microfluidic device based on a nanoshearing method [36]. While all of these methods have demonstrated suitable analytical performances, their application is still limited because of extensive and laborious protocols. The use of screen-printed electrodes (SPE) as the biosensor's transducer, without any surface modification prior to the functionalization with the biorecognition element, would considerably simplify the methodology. To date, only one electrochemical immunosensor for HER2 detection without prior modification of the electrode surface has been published [37]. In this work, a sandwich assay with enzymatic labelling allowed to achieve a low detection limit (4 ng/mL). However, a total assay time of 8 h revealed to be its major disadvantage because it is incompatible with a POC sensing strategy. Nanoparticle-based signal amplification has attracted considerable interest in the development of electrochemical methods. Distinct signal amplification methodologies can be achieved using nanomaterials. Their application as labels can greatly improve the signal transduction and simplify the detection strategy. Quantum Dots (QDs) revealed to be promising candidates as such labels, since they can be conjugated to antibodies and other proteins [38–41]. In addition, they can be synthesized with different compositions or with distinct core-shell structures, which can be useful for multiplexed sensing. The commonly used QDs are composed of a CdS or CdSe core with an external shell to provide functional groups for bioreceptor immobilization with inert and biocompatible coatings [38–40]. Comparing to laborious enzymatic methodologies, the use of QDs eliminates the need for substrate addition, which can contribute to the reduction of the analysis time by applying a straightforward process consisting of: QD dissolution to release the metal ions, electrochemical deposition (preconcentration) of the released ions and a potential (stripping) scan to detect the deposited metal. The obtained electrochemical signal can then quantitatively be related to the analyte concentration. Furthermore, working with electroactive labels also surpasses thermal instability aspects inherent to the nature of enzymes, the main difficulties in their use as labels [42,43]. To the best of our knowledge, this is the first electrochemical immunosensing strategy based on QDs as electrochemical label for in situ detection of HER2-ECD.

2.2. Reagents and solutions Rabit IgG monoclonal anti-human-HER2-ECD (clone 002) antibody (capture antibody – Ab-C), mouse IgG2a monoclonal biotinylated antihuman-HER2-ECD (clone 8B5DAC1) antibody (detection antibody – Ab-D), and a recombinant human HER2/ErBb2 protein (antigen) were obtained from Sino Biological Inc. Qdot® 655 streptavidin conjugate (QD-Strep) was purchased from Invitrogen - ThermoFisher Scientific. Bismuth ICP standard, acetic acid and sodium hydroxide were acquired from Merck. Human serum (from male AB clotted whole blood), albumin from bovine serum (BSA), nitric acid (HNO3) and tris(hydroxymethyl)aminomethane (Tris) were obtained from Sigma-Aldrich. Working solutions of BSA, the antibodies, antigen and QD-Strep were prepared in 0.1 M Tris-HNO3 pH 7.2 buffer (Tris Buffer). The Bi (III) solution (1 mg/L) was prepared in acetate buffer (0.1 M, pH 4.5). Ultra-pure water was obtained from a Millipore (Simplicity 185) water purification system. Buffers and solutions were prepared daily. The male human serum was stored at −20 °C. Serum samples (diluted 1:1 in Tris buffer) were spiked with HER2-ECD at different concentrations and analysed without further treatments. To evaluate the selectivity of the sensor, non-target proteins (possible interferents) were added to the serum solution. 2.3. Immunosensor development and detection strategy Scheme 1 elucidates the immunosensor's construction and detection strategy according to the following optimized protocol: (i) the Ab-C was immobilized on the SPCE surface by drop-casting a 10μL aliquot of a 25-μg/mL Ab-C solution, which was left overnight in a humidified chamber at 4 °C. Then, the SPCE was washed with Tris buffer before the (ii) incubation with BSA ((2% (m/V), 40 μL), for 30 min. After the free surface sites were efficiently blocked, (iii) 40 μL of a previously prepared mixture (10 min before use) containing the Ab-D (2 μg/mL), HER2-ECD and BSA 0.5% (m/V), was placed on the SPCE for 60 min. Afterwards, the washing step was repeated with Tris buffer and (iv) a 30-μL aliquot of QDs-Strep solution (5 nM, containing BSA 0.5% (m/V)) was placed on the sensor surface and left to incubate during 30 min. Prior to the measurements, a last washing step was performed with ultra-pure water. The analytical signal was obtained by differential pulse anodic stripping voltammetry (DPASV) based on a procedure described elsewhere [44]. Briefly, (v) 5 μL of HCl (1.0 M) was added to fully cover the working electrode and to release Cd2+ from the QDs. This was followed by the addition of 40 μL of an acetate buffer (0.1 M, pH 4.5) containing Bi(III) (1.0 mg/L). To activate the working electrode, a constant potential of +1.00 V was applied during 60 s. Then, the released cadmium ions were pre-concentrated by applying a potential of −1.10 V for 300 s. At the same time a bismuth film was formed. Finally, the potential was swept from −1.0 V to −0.7 V to strip the cadmium into the solution, recording the analytical signal (DPV parameters: pulse amplitude: 0.05 V; step potential: 0.01 V; modulation time: 0.01 s; interval time: 0.1 s).

2. Material and methods

3. Results and discussion

2.1. Apparatus and electrodes

3.1. Optimization of experimental conditions

Electrochemical measurements were carried out with a potentiostat/galvanostat (Autolab PGSTAT204, Metrohm Autolab) controlled by the NOVA software package v.1.10 (Metrohm Autolab). Disposable screen-printed carbon electrodes (with a 4-mm working electrode, a silver pseudoreference electrode and a carbon counter electrode, all made of conducting ink (SPCE, DRP-110)) as well the specific connector to interface the electrodes (DRP-CAC) were supplied by Metrohm DropSens.

Non-invasive analysis of cancer biomarkers can be performed in biological fluids such as serum. Bearing in mind the complex matrices of biological samples, it is mandatory to ensure that background interferences, due to the nonspecific adsorption of biomolecules, are minimized. Hence, the blockage of nonspecific adsorptions on the sensor platform after the modification with the Ab-C is of great importance for efficient and specific biomarker detection. The Ab-C 2

Talanta 208 (2020) 120430

M. Freitas, et al.

Scheme 1. Representation of the immunosensor construction and detection strategy.

concentration (25 μg/mL) was selected in accordance with our previous study [18] and was used throughout the work. Casein (2% m/V) and bovine serum albumin (BSA) (2% m/V) were the two tested blocking agents. When casein was used as the blocking agent no electrochemical signal was obtained. This was possibly due to an excessive blocking effect (Fig. S1 I). When BSA was used this behaviour was not verified. Nevertheless, and as can be seen in Fig. S1 (II), the difference between the blank signal and the signal obtained for the analyte was very low. Thus, BSA was added in other steps of the assay: (III) BSA 0.5% (m/V) in both the HER2-ECD and the Ab-D solutions and (IV) BSA 0.5% (m/V) in the HER2-ECD- and 1% (m/V) in the Ab-D solution. The obtained peak current intensities (ip) shown in Fig. S1 demonstrated that alternative III efficient blocked nonspecific adsorption, leading to the highest signal-to-blank ratio. Promising new approaches to improve the detection strategy in the development of electrochemical immunosensors have been aligned with nanotechnology through the use of nanolabels, such as QDs. Moreover, the functionalization of these nanomaterials with biomolecules such as streptavidin allows a strong binding to biotinylated antibodies. To optimize the conjugation between the biotinylated Ab-D and the QD-streptavidin conjugate (QD-Strep), different concentrations of Ab-D were tested (1, 2 and 4 μg/mL). The obtained ip values are presented in Fig. 1, and although this value increased significantly between 1 and 2 μg/mL, only a slightly increase was observed between 2 and 4 μg/mL, which indicates that the latter Ab-D concentration is close to saturation. Therefore, 2 μg/mL was chosen as the best compromise between the Ab-D concentration and the signal-to-blank ratio. In this work QDs were used to enhance the electrochemical detection, to simplify the methodology and to reduce the assay time. Thus, after the optimization of the surface blocking and the antibodies’ concentrations, three different QD concentrations were tested: 5, 10 and 20 nM (in terms of QDs) (Fig. 2A). The ip increased with the increase of the QD concentration both in the absence and presence of the analyte (50 ng/mL). Accordingly, 5 nM was selected because it provided the highest signal-to-blank ratio. BSA 0.5% (m/V) was added to the QDs solution to further minimize nonspecific adsorption, which also led to a much higher signal-to-blank ratio and a better precision of the results (Fig. 2B). Regarding the optimization of the assay, several formats were evaluated: step-by-step assays (A, B, C, D) and combined assays (E, F, G). The tested alternatives were: (A) antigen 60 min, Ab-D 60 min, QD

Fig. 1. Optimization of Ab-D concentration (1; 2 and 4 μg/mL). Experimental conditions: BSA (2% (m/V), Ab-C 25 μg/mL, HER2-ECD (0 and 50 ng/mL), QD 5 nM.

60 min (control assay, used during optimization studies), (B) antigen 60 min, Ab-D 60 min, QD 30 min, (C) antigen 30 min, Ab-D 60 min, QD 30 min, (D) antigen 60 min, Ab-D 30 min, QD 30 min; and the combined steps, which involve the pre-incubation of reagents: (E) antigen + Ab-D 60 min, QD 60 min, (F) antigen + Ab-D 60 min, QD 30 min, (G) antigen 60 min, Ab-D + QD 60 min (Fig. 3). The alternatives B and F provided the best signal-to-blank ratios. In both assays, 3

Talanta 208 (2020) 120430

M. Freitas, et al.

Fig. 2. (A) Optimization of QDs concentration (5; 10 and 20 nM) and (B) Current intensities obtained with QDs 5 nM in the absence and in the presence of BSA (0.5% (m/V)) in the solution. Experimental conditions: BSA (2% (m/V)), Ab-C (25 μg/mL), HER2-ECD (0 and 50 ng/mL), Ab-D (2 μg/mL).

3.2. Analytical performance for HER2-ECD determination in human serum The analytical performance of the developed immunosensor was tested using the previous optimized parameters. Initially, distinct HER2-ECD concentrations, in buffer solution, were analysed and a linear range was established between 5 and 150 ng/mL (Fig. S2). Then, male human serum samples (diluted 1:1 in Tris buffer) were spiked with HER2-ECD from 5.0 to 400 ng/mL. A linear relationship between the analyte concentration and ip was found between 10 and 150 ng/mL, according to the following equation: ip = (5.07 ± 0.14) [HER2ECD] + (18.4 ± 11.8), r = 0.9989, n = 5. The corresponding calibration plot as well typical voltammograms within the linear range are presented in Fig. 4. When comparing the calibration plots constructed in buffer solution and serum, a clear matrix effect was observed; the slope of the calibration plot in serum was 5 times lower than the slope obtained with the measurements in buffer. This could be due to human serum albumin, which is the most abundant protein in human blood, and globulins, especially immunoglobulins G (IgG) [45,46]. To avoid excessive blocking of the transducer's surface, BSA was not added to the antigen - Ab-D solution. The limits of detection (LOD) and quantification (LOQ) were calculated as the concentration corresponding to 3 × Sblank/m for the LOD and 10 × Sblank/m for the LOQ (Sblank: standard deviation of the blank signal; m: slope of the calibration plot). The obtained limit of detection (2.1 ng/mL) was far below the cut-off value established for HER2-ECD (15 ng/mL), which corroborates the adequacy and practical utility of the developed sensor. Table 1 summarizes the figures of merit of the developed sensor.

Fig. 3. Influence of the incubation time for step-by-step assay (A, B, C and D), and joining steps assay: HER2-ECD + Ab-D (E and F) and Ab-D + QDs (G). Experimental conditions: BSA (2% (m/V), Ab-C (25 μg/mL), HER2-ECD (25 ng/ mL), Ab-D (2 μg/mL), QDs (5 nM + BSA 0.5% (m/V)).

the QD incubation time was 30 min, which resulted in a reduction of both the blank and analytical signal and an increase of the signal-toblank ratio. The sensor's response to increasing concentrations of HER2ECD was assessed for both alternatives (B and F), obtaining similar sensitivities. Therefore, alternative F was chosen to proceed with the studies since the total time assay (120 min) was 60 min shorter than the one of alternative B. The combination of two steps not only allowed the increase of the precision of the results but also led to a more userfriendly assay.

3.3. Selectivity, stability, precision and recovery studies The selectivity of the immunosensor was tested through the analysis of other proteins that could be found in serum. In this study, the sensor's response towards cancer antigen 15–3, another breast cancer protein (CA15-3, 50 U/mL) and cystatin C, a biomarker of kidney function (550 ng/mL) was tested. Blank and HER2-ECD (50 ng/mL) solutions 4

Talanta 208 (2020) 120430

M. Freitas, et al.

Fig. 4. (A) Calibration plot using the developed immunosensor in the presence of growing concentrations of HER2-ECD in human serum and (B) examples of typical differential pulse voltammograms obtained within the established linear range (0, 10, 25, 50, 100 and 150 ng/mL), Experimental conditions: Ab-capture: 25 μg/mL, BSA: 2% (m/V), Ab-D: 2 μg/mL, QDs: 5 nM with addition of BSA 0.5% (m/V)). Table 1 Figures of merit of the developed biosensor for the analysis of the cancer biomarker HER2-ECD in Human Serum samples. Figure of merit Concentration interval (ng/mL) Correlation coefficient (r) Slope (m) (nA/(ng/mL)) Standard deviation of the slope (Sm) (nA/(ng/mL)) Intercept (b) (nA) Standard deviation of the intercept (Sa) (nA) Standard deviation of the linear regression (Sy/x) Standard deviation of the method (Sx0) Coefficient of variation of the method (Vx0) (%) Limit of detection (LOD) (ng/mL) Limit of quantification (LOQ) (ng/mL)

10–150 0.9989 5.07 0.14 18.4 11.8 16.1 3.2 5.6 2.1 7.1

were also assayed for comparison proposes. All the proteins were added to male human serum and their concentrations were chosen based on the values that can be expected in real clinical environments. The obtained ip values are presented in Fig. 5. In comparison to the analytical signal obtained for HER2-ECD, the other proteins did not show a significant response; the obtained ip values were very similar to the blank signal, which confirmed the sensor's selectivity towards HER2-ECD. The stability of the immunosensor was also studied. The as-prepared sensor was stored at 4 °C and measurements were performed during a month (Fig. S3). It was concluded that the sensor was stable for up to a week. So, the sensing phase of the proposed biosensor should be prepared on a weekly basis to guarantee a sensitive analysis. The intermediate precision and the reproducibility of the results were assessed by analyzing a 50-ng/mL HER2-ECD solution (in human serum) on the same day and on three different days, obtaining relative standard deviation (RSD) of 7.1% and 4.9%, respectively, which indicates that the optimized sensor provided precise results.

Fig. 5. Evaluation of the selectivity of the HER2-ECD sensor against other serum proteins. Experimental conditions: Ab-C: 25 μg/mL, BSA: 2% (m/V), AbD: 2 μg/mL, QDs: 5 nM with addition of BSA 0.5% (m/V)). Serum proteins concentration: HER2-ECD: 50 ng/mL, CA 15–3: 50 U/mL, Cystatin C: 550 ng/ mL.

5

6

SPCE/MWCNT-AuNP

FTO/NFG/AgNP/PANI GCE/Fe3O4-APTMS CILE-MWCNT/AuNP SPCE/AuNP

AuE/AuNP-MPA-Cys GCE/poly-DPB(AuNP) NEEs SPCE/MBs SPCE/MBs

SPE 8 × SPE/Strep-MBs or ProtA-MBs

HER2-ECD

SK-BR-3 HER2 HER2 HER2-ECD

HER2 HER2 HER2 HER2 ErbB2

HER2 HER2

1h ~15 min

> 22 h >4h n.d. ~15 min ~40 min

>4h >3h > 30 h ~10 min

~15 min

–

Transducer preparation timea

1h ~12 h 45 min

~12 h ~12 h 2h 40 min 1h

~12 h 2h 6h ~12 h

~12 h

~12 h

Modification with the biorecognition elementa

3 weeks 1 week

3 weeks n.d. n.d. 10 days n.d.

45 days 10 days n.d. n.d.

n.d.

7 days

Long-term stability

HRP AP

n.a. Hydrazin HRP AP HRP

n.a. Hydrazin n.a. AP

CdSe@ZnS QDs AP

Label

Amperometry DPV

DPV SWSV CV DPV Amperometry

DPV DPV EIS LSV

LSV

DPV

Technique

30 min 1 h 10 min 7 h 10 min 2 h 05 min 1h

[Fe (CN)6]3−/4− Silver MB 1-NP HQ

22 min 2 h 51 min 1 h 51 min

30 min 2h 35 min 2 h 50 min

[Fe (CN)6]3−/4− Silver [Fe (CN)6]3−/4− Silver

HQ 1-NP

2 h 20 min

2h

Assay time

Silver

Cadmium

Detection

1 μg/mL 1.8, 2.6 and 3.4 ng/mL

0.995 pg/mL 0.037 pg/mL 40 ng/mL 6.0 ng/mL 26 pg/mL

2 cells/mL 2.0 × 10−5 ng/mL 7.2 ng/mL 4.4 ng/mL

0.16 ng/mL

2.1 ng/mL

LOD

Spiked human serum Spiked human serum Whole blood Patient serum Patient serum Spiked human serum Patient serum Cancer cells Cell lysates Patient serum Patient serum and cell lysates Cell lysates Spiked human serum

Sample

[28] [29]

[23] [24] [25] [26] [27]

[19] [20] [21] [22]

This study [18]

Ref

1-NP – 1-naphthol; AgNP – silver nanoparticles; AP – alkaline phosphatase; APTMS – (3-Aminopropyl)triethoxysilane; AuE – gold electrode; AuNPs – gold nanoparticles; Cys – cysteamine; CV – cyclic voltammetry; CILE –carbon ionic liquid electrode; FTO – fluorine doped tin oxide; GCE – glassy carbon electrode; DPB – 2,5-bis(2-thienyl)-1H-pyrrole-1-(p-benzoic acid); DPV – differential pulse voltammetry; HER2 – human epidermal growth factor receptor 2; HRP – horseradish peroxidase; HQ – hydroquinone; LSV – linear sweep voltammetry; MBs – magnetic beads; MB – methylene blue; MPA – 3-mercaptopropionic acid; MWCNT – multiwalled carbon nanotube; NEEs – nanoelectrode ensembles; NFG – nitrogen-doped graphene; n.d. – no data; n.a. – not applicable; PANI – polyaniline; QDs – quantum dots; SPCE – screen-printed carbon electrode; SWASV – square wave anodic stripping voltammetry; SWV – Square wave voltammetry. a Overnight incubations were considered as a 12-h period for comparison purposes.

Bare SPCE

Transducer

Sensing surface

HER2-ECD

Breast cancer analyte

Table 2 Summary of experimental parameters of electrochemical immunoassay-based procedures for HER2 analysis.

M. Freitas, et al.

Talanta 208 (2020) 120430

Talanta 208 (2020) 120430

M. Freitas, et al.

To demonstrate the potential clinical utility of the immunosensor, recovery studies were performed using serum samples spiked with three different HER2-ECD concentrations: 25, 75 and 125 ng/mL. The average recoveries were found to be 105.6, 105.0 and 103.5% with RSDs of 5.6, 5.0 and 3.5%, respectively (n = 3). This indicates that the developed sensor provided both precise and accurate results.

financially supported by FCT through the doctoral research Grant financed by fellowship SFRH/BD/111942/2015. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.talanta.2019.120430.

3.4. Comparison with previous reported electrochemical immunosensors and immunoassays

References

The proposed immunosensor was compared with previously published immunosensors and immunoassays with electrochemical detection for HER2 analysis. As can be seen in Table 2, distinct configurations with comparable performances were described. The immunosensor developed in this work has a competitive assay time that is only surpassed by the label-free sensors [19,21,23] and by three works in which an enzymatic label was employed [24,27,28]. However, some of these shorter assay times are preceded by complex modifications of the electrode surface, as well as time-consuming procedures, which appears as a major drawback [19,21,23,24]. Moreover, the work developed by Patris et al. presented a good compromise between assay time and LOD, considering the complexity of the sample (cell lysates) [28]. The electrochemical magneto-immunoassay described by Eletxigerra et al. [27] demonstrated an exceptional LOD and assay time. Nevertheless, the dependence on an enzymatic substrate and the production of a substantial amount of waste, due to the volume of mediator needed for the measurement step, are the major drawbacks of the reported work. The remaining works did not improve the assay time of our assay. Accordingly, the immunosensor developed in this work presents a user-friendly straightforward 3-step assay protocol, within a highly competitive assay time, and achieving a low LOD (< reference cut-off value). Furthermore, an additional overview of the use of metallic and carbon-based QDs for the immunosensing of different breast cancer biomarkers is summarized in Table SI 1. In some works, the QDs were used as detection label while in others they were employed in the modification of the transducer surface. Most of these works imply laborious protocols [38,40,47,48] and longer assay times [41,49]. Additionally, our immunosensor is the first one that employs QDs for the development of an electrochemical immunosensor for HER2-ECD.

[1] F. Bray, J. Ferlay, I. Soerjomataram, R.L. Siegel, L.A. Torre, A. Jemal, Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries, CA Cancer J. Clin. 68 (2018) 394–424, https://doi.org/10. 3322/caac.21492. [2] J. Ferlay, I. Soerjomataram, R. Dikshit, S. Eser, C. Mathers, M. Rebelo, D.M. Parkin, D. Forman, F. Bray, Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012, Int. J. Cancer 136 (2015) E359–E386, https://doi.org/10.1002/ijc.29210. [3] E. Altobelli, A. Lattanzi, R. Paduano, G. Varassi, F. di Orio, Colorectal cancer prevention in Europe: burden of disease and status of screening programs, Prev. Med. 62 (2014) 132–141, https://doi.org/10.1016/j.ypmed.2014.02.010. [4] E. Altobelli, F. D'Aloisio, P.M. Angeletti, Colorectal cancer screening in countries of European Council outside of the EU-28, World J. Gastroenterol. 22 (2016) 4946–4957, https://doi.org/10.3748/wjg.v22.i20.4946. [5] G. Ronco, J. Dillner, K.M. Elfström, S. Tunesi, P.J.F. Snijders, M. Arbyn, H. Kitchener, N. Segnan, C. Gilham, P. Giorgi-Rossi, J. Berkhof, J. Peto, C.J.L.M. Meijer, J. Cuzick, M. Zappa, F. Carozzi, M. Confortini, P. Dalla Palma, M. Zorzi, A. Del Mistro, A. Gillio-Tos, C. Naldoni, D. Rijkaart, F. Van Kemenade, N. Bulkmans, D. Heideman, R. Rozendaal, G. Kenter, M. Almonte, C. Roberts, M. Desai, A. Sargent, W. Ryd, P. Naucler, Efficacy of HPV-based screening for prevention of invasive cervical cancer: follow-up of four European randomised controlled trials, Lancet 383 (2014) 524–532, https://doi.org/10.1016/S01406736(13)62218-7. [6] E. Altobelli, A. Lattanzi, Cervical carcinoma in the European Union, Int. J. Gynecol. Cancer 25 (2015) 474–483, https://doi.org/10.1097/igc.0000000000000374. [7] E. Altobelli, L. Rapacchietta, P.M. Angeletti, L. Barbante, F.V. Profeta, R. Fagnano, Breast cancer screening programmes across the WHO European region: differences among countries based on national income level, Int. J. Environ. Res. Public Health 14 (2017), https://doi.org/10.3390/ijerph14040452. [8] International Agency for Research on Cancer, Cancer Screening in the European Union: Report on the Implementation of the Council Recommendation on Cancer Screening, (2017). [9] J. Ferlay, M. Colombet, I. Soerjomataram, T. Dyba, G. Randi, M. Bettio, A. Gavin, O. Visser, F. Bray, Cancer incidence and mortality patterns in Europe: estimates for 40 countries and 25 major cancers in 2018, Eur. J. Cancer 103 (2018) 356–387, https://doi.org/10.1016/j.ejca.2018.07.005. [10] M.J.M. Broeders, P. Allgood, S.W. Duffy, S. Hofvind, I.D. Nagtegaal, E. Paci, S.M. Moss, L. Bucchi, The impact of mammography screening programmes on incidence of advanced breast cancer in Europe: a literature review, BMC Canc. 18 (2018) 860, https://doi.org/10.1186/s12885-018-4666-1. [11] M.J. Duffy, N. Harbeck, M. Nap, R. Molina, A. Nicolini, E. Senkus, F. Cardoso, Clinical use of biomarkers in breast cancer: updated guidelines from the european group on tumor markers (EGTM), Eur. J. Cancer 75 (2017) 284–298, https://doi. org/10.1016/j.ejca.2017.01.017. [12] A.C. Wolff, M. Elizabeth, H. Hammond, K.H. Allison, B.E. Harvey, P.B. Mangu, J.M.S. Bartlett, M. Bilous, I.O. Ellis, P. Fitzgibbons, W. Hanna, R.B. Jenkins, M.F. Press, P.A. Spears, G.H. Vance, G. Viale, L.M. Mcshane, M. Dowsett, Human epidermal growth factor receptor 2 testing in breast cancer: American society of clinical oncology/college of American pathologists clinical practice guideline focused update, J. Clin. Oncol. 36 (2018) 2105–2122, https://doi.org/10.1200/JCO. [13] F.B. De Abreu, G.N. Schwartz, W.A. Wells, G.J. Tsongalis, Personalized therapy for breast cancer, Clin. Genet. 86 (2014) 62–67, https://doi.org/10.1111/cge.12381. [14] C. Tsé, A.S. Gauchez, W. Jacot, P.J. Lamy, HER2 shedding and serum HER2 extracellular domain: biology and clinical utility in breast cancer, Cancer Treat Rev. 38 (2012) 133–142, https://doi.org/10.1016/j.ctrv.2011.03.008. [15] A. Perrier, J. Gligorov, G. Lefèvre, M. Boissan, The extracellular domain of Her2 in serum as a biomarker of breast cancer, Lab. Investig. 98 (2018) 696–707, https:// doi.org/10.1038/s41374-018-0033-8. [16] M.A. Huaman, C.T. Fiske, T.F. Jones, J. Warkentin, B.E. Shepherd, F. Maruri, T.R. Sterling, Exercise altered the skeletal muscle MicroRNAs and gene expression profiles in burn rats with hindlimb unloading, J. Burn Care Res. 143 (2015) 951–959, https://doi.org/10.1017/S0950268814002131.Tuberculosis. [17] I.E. Tothill, Biosensors for cancer markers diagnosis, Semin. Cell Dev. Biol. 20 (2009) 55–62, https://doi.org/10.1016/j.semcdb.2009.01.015. [18] M. Freitas, H.P.A. Nouws, C. Delerue-Matos, Electrochemical sensing platforms for HER2-ECD breast cancer biomarker detection, Electroanalysis 31 (2019) 121–128, https://doi.org/10.1002/elan.201800537. [19] R. Salahandish, A. Ghaffarinejad, S.M. Naghib, K. Majidzadeh-A, H. Zargartalebi, A. Sanati-Nezhad, Nano-biosensor for highly sensitive detection of HER2 positive breast cancer, Biosens. Bioelectron. 117 (2018) 104–111, https://doi.org/10.1016/ j.bios.2018.05.043. [20] M. Shamsipur, M. Emami, L. Farzin, R. Saber, A sandwich-type electrochemical immunosensor based on in situ silver deposition for determination of serum level of

4. Conclusions A novel and efficient electrochemical immunosensor for the analysis of the breast cancer biomarker HER2-ECD, with a total time assay of 2 h, was developed. This work highlights the simplicity of the assay, with an actual hands-on-time of less than 30 min, without resorting to laborious electrode surface modifications. An electroactive QD label was employed and a limit of detection of 2.1 ng/mL was achieved, corresponding to the detection of 1.18 fmol of the analyte (sample volume = 40 μL). The analytical performance was tested in spiked human serum samples, demonstrating an excellent performance in a wide linear range with a high sensitivity, stability, precision and accuracy. The applicability and selectivity of the proposed methodology was tested and confirmed through the analysis of distinct serum proteins in human serum. Acknowledgements The authors are grateful for the financial support from the Fundação para a Ciência e a Tecnologia (FCT)/the Ministério da Ciência, Tecnologia e Ensino Superior (MCTES) through national funds (Portugal) (UID/QUI/50006/2019). This work was also supported by the European Union through projects Norte-01-0145-FEDER-000024 and Norte-01-0145-FEDER-000011, co-funded by FEDER in the scope of CCDR-N and NORTE2020 Partnership Agreement. Maria Freitas is 7

Talanta 208 (2020) 120430

M. Freitas, et al.

[21]

[22]

[23]

[24] [25] [26] [27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35] X. Li, C. Shen, M. Yang, A. Rasooly, Polycytosine DNA electric-current-generated immunosensor for electrochemical detection of human epidermal growth factor receptor 2 (HER2), Anal. Chem. 90 (2018) 4764–4769, https://doi.org/10.1021/ acs.analchem.8b00023. [36] R. Vaidyanathan, S. Rauf, M.J.A. Shiddiky, M. Trau, Tuneable surface shear forces to physically displace nonspecific molecules in protein biomarker detection, Biosens. Bioelectron. 61 (2014) 184–191, https://doi.org/10.1016/j.bios.2014.03. 061. [37] S.D. Tallapragada, K. Layek, R. Mukherjee, K.K. Mistry, M. Ghosh, Development of screen-printed electrode based immunosensor for the detection of HER2 antigen in human serum samples, Bioelectrochemistry 118 (2017) 25–30, https://doi.org/10. 1016/j.bioelechem.2017.06.009. [38] X. Wu, T. Xiao, Z. Luo, R. He, Y. Cao, Z. Guo, W. Zhang, Y. Chen, A micro-/nanochip and quantum dots-based 3D cytosensor for quantitative analysis of circulating tumor cells, J. Nanobiotechnol. 16 (2018) 65, https://doi.org/10.1186/s12951018-0390-x. [39] K. Boriachek, M.N. Islam, V. Gopalan, A.K. Lam, N.T. Nguyen, M.J.A. Shiddiky, Quantum dot-based sensitive detection of disease specific exosome in serum, Analyst 142 (2017) 2211–2219, https://doi.org/10.1039/c7an00672a. [40] J.A.A. Ho, Y.C. Lin, L.S. Wang, K.C. Hwang, P.T. Chou, Carbon nanoparticle-enhanced immunoelectrochemical detection for protein tumor marker with cadmium sulfide biotracers, Anal. Chem. 81 (2009) 1340–1346, https://doi.org/10.1021/ ac801832h. [41] Y. Luo, Y. Wang, H. Yan, Y. Wu, C. Zhu, D. Du, Y. Lin, SWCNTs@GQDs composites as nanocarriers for enzyme-free dual-signal amplification electrochemical immunoassay of cancer biomarker, Anal. Chim. Acta 1042 (2018) 44–51, https://doi. org/10.1016/j.aca.2018.08.023. [42] Y. Liu, L. Zhang, Q. Guo, H. Hou, T. You, Enzyme-free ethanol sensor based on electrospun nickel nanoparticle-loaded carbon fiber paste electrode, Anal. Chim. Acta 663 (2010) 153–157, https://doi.org/10.1016/j.aca.2010.01.061. [43] M. Freitas, H.P.A. Nouws, C. Delerue-Matos, Electrochemical biosensing in cancer diagnostics and follow-up, Electroanalysis 30 (2018) 1576–1595, https://doi.org/ 10.1002/elan.201800193. [44] D. Martín-Yerga, M.B. González-García, A. Costa-García, Electrochemical immunosensor for anti-tissue transglutaminase antibodies based on the in situ detection of quantum dots, Talanta 130 (2014) 598–602, https://doi.org/10.1016/j. talanta.2014.07.010. [45] J.T. Busher, Serum albumin and globulin, in: H.K. Walker, W.D. Hall, J.W. Hurst (Eds.), Clinical Methods: the History, Physical, and Laboratory Examinations, third ed., Butterworths, Boston (USA), 1990(Chapter 101). [46] S.E.F. Melanson, M.J. Tanasijevic, P. Jarolim, Cardiac troponin assays: a view from the clinical chemistry laboratory, Circulation 116 (2007) e501–e504, https://doi. org/10.1161/CIRCULATIONAHA.107.722975. [47] M. Hasanzadeh, S. Rahimi, E. Solhi, A. Mokhtarzadeh, N. Shadjou, J. Soleymani, S. Mahboob, Probing the antigen-antibody interaction towards ultrasensitive recognition of cancer biomarker in adenocarcinoma cell lysates using layer-by-layer assembled silver nano-cubics with porous structure on cysteamine caped GQDs, Microchem. J. 143 (2018) 379–392, https://doi.org/10.1016/j.microc.2018.08. 028. [48] M. Hasanzadeh, S. Tagi, E. Solhi, N. Shadjou, A. Jouyban, A. Mokhtarzadeh, Immunosensing of breast cancer prognostic marker in adenocarcinoma cell lysates and unprocessed human plasma samples using gold nanostructure coated on organic substrate, Int. J. Biol. Macromol. 118 (2018) 1082–1089, https://doi.org/10. 1016/j.ijbiomac.2018.06.091. [49] M. Hasanzadeh, H.N. Baghban, N. Shadjou, A. Mokhtarzadeh, Ultrasensitive electrochemical immunosensing of tumor suppressor protein p53 in unprocessed human plasma and cell lysates using a novel nanocomposite based on poly-cysteine/graphene quantum dots/gold nanoparticle, Int. J. Biol. Macromol. 107 (2018) 1348–1363, https://doi.org/10.1016/j.ijbiomac.2017.11.006.

HER2 in breast cancer patients, Biosens. Bioelectron. 103 (2018) 54–61, https:// doi.org/10.1016/j.bios.2017.12.022. E. Arkan, R. Saber, Z. Karimi, M. Shamsipur, A novel antibody-antigen based impedimetric immunosensor for low level detection of HER2 in serum samples of breast cancer patients via modification of a gold nanoparticles decorated multiwall carbon nanotube-ionic liquid electrode, Anal. Chim. Acta 874 (2015) 66–74, https://doi.org/10.1016/j.aca.2015.03.022. R.C.B. Marques, S. Viswanathan, H.P.A. Nouws, C. Delerue-Matos, M.B. GonzálezGarcía, Electrochemical immunosensor for the analysis of the breast cancer biomarker HER2 ECD, Talanta 129 (2014) 594–599, https://doi.org/10.1016/j. talanta.2014.06.035. M. Emami, M. Shamsipur, R. Saber, R. Irajirad, An electrochemical immunosensor for detection of a breast cancer biomarker based on antiHER2–iron oxide nanoparticle bioconjugates, Analyst 139 (2014) 2858–2866, https://doi.org/10.1039/ c4an00183d. Y. Zhu, P. Chandra, Y.B. Shim, Ultrasensitive and selective electrochemical diagnosis of breast cancer based on a hydrazine-Au nanoparticle-aptamer bioconjugate, Anal. Chem. 85 (2013) 1058–1064, https://doi.org/10.1021/ac302923k. S.P. Mucelli, M. Zamuner, M. Tormen, G. Stanta, P. Ugo, Nanoelectrode ensembles as recognition platform for electrochemical immunosensors, Biosens. Bioelectron. 23 (2008) 1900–1903, https://doi.org/10.1016/j.bios.2008.02.027. Q.A.M. Al-Khafaji, M. Harris, S. Tombelli, S. Laschi, A.P.F. Turner, M. Mascini, G. Marrazza, An electrochemical immunoassay for HER2 detection, Electroanalysis 24 (2012) 735–742, https://doi.org/10.1002/elan.201100501. U. Eletxigerra, J. Martinez-Perdiguero, S. Merino, R. Barderas, R.M. TorrenteRodríguez, R. Villalonga, J.M. Pingarrón, S. Campuzano, Amperometric magnetoimmunosensor for ErbB2 breast cancer biomarker determination in human serum, cell lysates and intact breast cancer cells, Biosens. Bioelectron. 70 (2015) 34–41, https://doi.org/10.1016/J.BIOS.2015.03.017. S. Patris, P. De Pauw, M. Vandeput, J. Huet, P. Van Antwerpen, S. Muyldermans, J.M. Kauffmann, Nanoimmunoassay onto a screen printed electrode for HER2 breast cancer biomarker determination, Talanta 130 (2014) 164–170, https://doi. org/10.1016/j.talanta.2014.06.069. H. Ilkhani, A. Ravalli, G. Marrazza, Design of an affibody-based recognition strategy for human epidermal growth factor receptor 2 (HER2) detection by electrochemical biosensors, Chemosensors 4 (2016) 23, https://doi.org/10.3390/ chemosensors4040023. J.G. Pacheco, P. Rebelo, M. Freitas, H.P.A. Nouws, C. Delerue-Matos, Breast cancer biomarker (HER2-ECD) detection using a molecularly imprinted electrochemical sensor, Sens. Actuators B Chem. 273 (2018) 1008–1014, https://doi.org/10.1016/j. snb.2018.06.113. E. Chocholova, T. Bertok, L. Lorencova, A. Holazova, P. Farkas, A. Vikartovska, V. Bella, D. Velicova, P. Kasak, A.A. Eckstein, J. Mosnacek, D. Hasko, J. Tkac, Advanced antifouling zwitterionic layer based impedimetric HER2 biosensing in human serum: glycoprofiling as a novel approach for breast cancer diagnostics, Sens. Actuators B Chem. 272 (2018) 626–633, https://doi.org/10.1016/j.snb.2018. 07.029. S. Sharma, J. Zapatero-Rodríguez, R. Saxena, R. O'Kennedy, S. Srivastava, Ultrasensitive direct impedimetric immunosensor for detection of serum HER2, Biosens. Bioelectron. 106 (2018) 78–85, https://doi.org/10.1016/j.bios.2018.01. 056. K. Malecka, D. Pankratov, E.E. Ferapontova, Femtomolar electroanalysis of a breast cancer biomarker HER-2/neu protein in human serum by the cellulase-linked sandwich assay on magnetic beads, Anal. Chim. Acta (2019), https://doi.org/10. 1016/j.aca.2019.05.052. S. Carvajal, S.N. Fera, A.L. Jones, T.A. Baldo, I.M. Mosa, J.F. Rusling, C.E. Krause, Disposable inkjet-printed electrochemical platform for detection of clinically relevant HER-2 breast cancer biomarker, Biosens. Bioelectron. 104 (2018) 158–162, https://doi.org/10.1016/j.bios.2018.01.003.

8